凡纳滨对虾丝氨酸蛋白酶基因(Lv-SP)的克隆及表达分析  

Molecular Cloning and Expression Analysis of Lv-SP Gene from Litopenaeus vannamei

作　　者：宋巧珍 邹枘峰 张亦陈[1] 耿绪云 刘逸尘[1] SONG Qiao-zhen;ZOU Rui-feng;ZHANG Yi-chen(Tianjin Key Laboratory of Animal and Plant Resistance/College of Life Science,Tianjin Normal University,Tianjin 300387)

机构地区：[1]天津市动植物抗性重点实验室/天津师范大学生命科学学院,天津300387 [2]天津市水生动物疫病预防控制中心,天津300221

出　　处：《安徽农业科学》2018年第12期102-106,181,共6页Journal of Anhui Agricultural Sciences

基　　金：国家自然科学基金项目(31472299);天津市应用基础与前沿技术研究计划(15JCZDJC33800);天津市水产生态及养殖重点实验室开放基金项目(TJAE2015005)

摘　　要：[目的]研究丝氨酸蛋白酶基因(Lv-SP)在凡纳滨对虾天然免疫应答过程中的作用。[方法]利用PCR技术克隆凡纳滨对虾丝氨酸蛋白酶基因(Lv-SP),并进行生物信息学分析,进一步通过实时荧光定量PCR(qRT-PCR)方法对其进行组织表达分析以及白斑杆状病毒(WSSV)刺激后的表达变化分析。[结果]成功从凡纳滨对虾体内克隆了Lv-SP基因的开放阅读框ORF,碱基数为1 104 bp,共编码367个氨基酸,在线分析软件预测其蛋白分子量为41 k D,等电点PI为6.93;在线分析软件预测显示:Lv-SP基因预测的氨基酸序列保守性很高,含有1个clip结构域和1个高度保守的SP结构域(Tryp-SPc);系统进化发生分析表明:Lv-SP氨基酸序列与中国明对虾以及中华绒螯蟹的丝氨酸蛋白酶(SP)同源性较高,而与按蚊、小蜂窝甲虫的SP同源性较低;qRT-PCR分析结果显示:Lv-SP基因在凡纳滨对虾不同组织中均有表达,并且在鳃和血中表达量较高,而在心和肝中表达量较低,病毒WSSV刺激后,Lv-SP基因在48.0 h和72.0 h有显著的上调表达趋势。[结论]Lv-SP基因保守性比较高,并且其在一定程度上参与了对虾应答病毒侵染的免疫反应,推测它有可能作为重要的免疫调控基因在对虾的防御应答过程中发挥作用。[Objective]Functional research of serine protease(SP)gene(Lv-SP)from Litopenaeus vannamei in innate immunity response.[Method]Lv-SP gene was cloned by PCR.The bioinformatics analysis was performed by online software.Expression profiles of Lv-SP gene in tissue and in response to WSSV challenge were analyzed by qRT-PCR.[Result]The length of the open reading frame(ORF)of Lv-SP gene contained 1 104 bp,encoding 367 amino acids.The predicted protein molecular weight and calculated isoelectric point was 41 kD and 6.93,respectively.Analyzed by online bioinformatics software,Lv-SP was highly conservative.It contained a clip domain and a highly conserved SP domain(Tryp-SPc).Phylogenetic analysis showed that comparing to the SP protein in Fenneropenaeus chinensis and Eriocheir sinensis.,Lv-SP had highest homologous.However,Lv-SP had lowest homology comparing to SP in Anopheles gambiae and Aethina tumida.qRT-PCR results showed that Lv-SP gene were expressed in different tissues of L.vannamei.Lv-SP gene had high expression level in gill and hemocytes and had low expression level in heart and hepatopancreas.The expression of Lv-SP was up-regulated significantly in 48.0 h and 72.0 h after WSSV infection.[Conclusion]Lv-SP has a typical clip domain and is highly conserved with other SPs with immunological functions.Lv-SP from L.vannamei might plays an important role in the immune response according to the infection of pathogen.

关 键 词：凡纳滨对虾 丝氨酸蛋白酶 基因克隆 免疫应答 WSSV刺激 

分 类 号：S96[农业科学—水产养殖]